1. Field of the Invention
The present invention relates to improvements in total internal reflection (TIR) spectroscopy, and more particularly to an apparatus and method for TIR-based spectroscopic imaging with improved angular scanning, particularly attenuated total reflection (ATR) imaging spectroscopy and microscopy.
2. Description of the Related Art
Attenuated total reflection (ATR) spectroscopy, also known as internal reflection spectroscopy (IRS), originates in the observation by Newton almost two centuries ago that a propagating wave of radiation which undergoes total internal reflection (TIR) in a higher index of refraction medium in contact with a lower index of refraction medium gives rise to an evanescent field in the lower refractive index medium. It was, however, not until 1960 that this phenomenon was exploited for producing absorption spectra. Since then, numerous ATR-based applications have been developed, many of them in the biosensor field. In biosensors based on ATR, the evanescent field probes a thin layer (penetration depth about one wavelength) of sample-containing material at a solid/liquid interface, resulting in a measurable attenuation of the reflected radiation in proportion to the imaginary part of the refractive index of the thin layer, i.e., absorption spectroscopy.
As is well-known, the spectrum in ATR-spectroscopy consists of a reflectance vs. wavelength curve or spectrum, where the angle of incidence may be scanned in order to vary evanescent wave penetration depth (see e.g., Harrick, N. J., Internal Reflection Spectroscopy, Harrick Scientific Corp., New York, 1967; and Mirabella, F. M. Jr., Internal Reflection Spectroscopy: Review and Supplement, Harrick Scientific Corp., New York, 1985). Photon absorbance at the ATR-sensor surface, with or without evanescent electrical field strength enhancing metal film or particles, is detected as a more or less sharp and deep minimum, or dip, in the internal reflectance (TIR) curve, and the depth (absorbance peak) is a measure of the amount of detected sample, and the wavelength is used to identify the kind of molecule.
A variant of ATR-spectroscopy, hereinafter referred to as SPR-spectroscopy, is based on surface plasmon resonance (SPR) in an electrical field-strengthening layer of metal film or metal islands or particles applied on the solid medium at the solid/liquid interface. The angle (or wavelength) corresponding to the minimum or centroid of the photon absorbance peak, or reflectance dip, and/or the change in angle, is a measure of the amount of detected sample. More particularly, the change in SPR-angle and/or SPR-wavelength is a measure of the change in the real part of the refractive index (n) and/or the change in the thickness (d) of the sample interaction layer or layers at the solid/liquid interface. If the sample also allows photon absorbance, this can be measured via changes in the shape (reflectance minimum and dip width) of the SPR-ATR-curve.
The surface concentration of the sample can be calculated from the shift in SPR-angle and/or SPR-wavelength by use of well-known relations between surface concentration and layer structural parameters n, d, and empirical or calculated refractive index increment of the sample solute (De Feijter, J. A., et al., Biopolymers 17:1759, (1978); and Salaman, Z., et al., Biochemistry 33:13706, (1994)).
In some SPR-spectroscopic optic configurations, e.g., using both p- and s-polarized light, the photon absorbance is converted into a peak rather than a dip in the reflectance curve, and the angle (or wavelength) position, or shift in position, for this peak is used as a quantitative measure of the amount, or change in amount, of detected sample.
SPR-based sensors are commercially available for use in research and development, for example the BIACORE(copyright) instrument line from Biacore AB, Uppsala, Sweden. These instruments use a sensor glass chip covered with a thin gold film and an integrated fluid cartridge for passing sample fluid and other fluids over the sensor chip. A fan shaped beam of light is coupled to the sensor chip via a prism such that an angular range of incident light is reflected internally along a line at the glass/gold film interface creating a plasmon evanescent field at the gold film/fluid interface, and the reflected light intensity distribution versus angle of incidence for a row of sensor spots along the illuminated line, is detected by a photodetector array.
The sensitivity in the detectable change of the angle (or wavelength) at the dip or peak (or in some cases, dips or peaks) of the SPR spectrum is mainly limited by the degree of constancy, drift and noise of the background light intensity of the TIR-curve. Ideally, the TIR-curve is constant versus angle. In practice, however, due to variation of reflectance with the angle of incidence, and the radiation distribution from the light source, the TIR-curve will generally be a gaussian type curving line with at least one maximum and which at its low and high angle ends, respectively, is more or less sloping. The sensitivity of SPR-spectroscopy is, of course, particularly in the case of kinetic studies (measurement of the time dependence of the reflectance curve, shape and/or position), higher the more stable the light intensity at total internal reflection is over the angular or wavelength range of interest, i.e. the less dependent the xe2x80x9cbackgroundxe2x80x9d TIR-curve is on the angle (or wavelength).
The sensitivity of SPR-spectroscopy can thus be further increased if the TIR-curve is normalized, such as by a computer software algorithm. This usually requires highly stable TIR-curve data, i.e. low temporal noise and drift, and that the TIR-curve has as little curvature and sloping as possible and is as smooth as possible. Such computerized normalization of the TIR-curve is, for example, done in the BIACORE(copyright) instruments mentioned above, where the normalization procedure is performed on the TIR-spectrum with a sample of refractive index high enough to give TIR for the angle range used. Due to the use of a focused light beam (spot or line) and a static optical system in the present BIACORE(copyright) instruments, the illuminated sensor surface for TIR is fixed (stationary). The stability of the provided irradiance is therefore only limited by noise and drift in the light source.
Whereas conventional ATR-spectroscopy measures the surface characteristics of a sample at a fixed small spot or a thin line, spatial scanning of the incident light beam across a surface area may produce an image of the spectral property distribution over the scanned area. This technique is usually referred to as ATR-microscopy. Thus, for instance, various arrangements for surface plasmon microscopy (SPM) have been proposed for detection of the spatial distribution of the refractive index. Rather than spatially scanning a focused line or light beam across the sensor surface area with time, it has also been described to momentarily illuminate and image the whole surface area in question and scan the incident light angle or wavelength with time. A biosensor based on ATR-microscopy may, for example, be used for analyzing a large number, or an array, of sample spots simultaneously.
An example of an ATR-sensor apparatus of the last-mentioned type based on scanning the angle of incidence of a probing collimated (parallel) beam which illuminates the whole surface area is disclosed in WO 98/34098 (the full disclosure of which is incorporated by reference herein). A representative illustration of this system is given in FIG. 1 herein, where a light source, LS, illuminates a collimator optics, CO, to produce a parallel light beam. The beam passes an interference filter, I, as a monochromatic beam and impinges on a first flat scanner mirror, SM1, to be deflected onto a second scanning mirror, SM2. The latter deflects the beam into a prism Pr for coupling the light into a sensor surface, SS, the beam being totally internally reflected at the sensor interface side of the coupling prism. The p-polarized component of the beam then passes a polarizer, P, whereupon the beam is directed into a spherical objective, SO, to produce a real image on a photo detector array or matrix, D, of the sensor surface area from its reflected light intensity pattern.
The detector array D is arranged such that the real image of the sensor area is produced on a rectangular part, D, of the array, the objective SO having its real image plane positioned at the plane of the photodetector array. Numerals 1, 2 and 3 at the detector array D denote the respective images of the corresponding subzones on the sensor surface SS, indicated at 1xe2x80x2, 2xe2x80x2 and 3xe2x80x2, respectively.
The two scanning mirrors SM1 and SM2 have a related rotational or oscillating movement to produce an angularly scanned collimated beam incident within a range of angles of incidence on the sensor surface side of the prism Pr. xe2x80x9cBeam walkingxe2x80x9d of the illuminated area, which is caused by refraction of the collimated light at the entrance of the prism Pr during the scan of the angle of entrance and would give an irregular irradiance (radiant power per unit area) at the sensor surface, is avoided by synchronizing the movements of the scanning mirrors SM1 and SM2 (as well as adapting the distances and angles between mirrors and prism, and scanned angular range of the mirrors) to provide a fixed center for the intersection of the incident collimated light beam with the sensor surface SS.
In the general type of ATR-microscope described above, the momentary incident angle of the collimated light beam irradiating the sensor surface during the angle scan may de determined by various means. WO 98/34098 proposes to determine the momentary incident angle by detecting a part of the light beam reflected at the sensor surface on a second detector. More particularly, the objective SO comprises an additional part having its back focal plane positioned at the plane of the photodetector array to permit a minor part of the reflected collimated beam to be focused onto a minor, linear part of the detector area so that each angle of reflection corresponds to a specific linear detector position within the detector area.
While in the ATR-microscope described in WO 98/34098 the fixed center of the illuminated area thus gives a more stable irradiance, or optical power distribution across the cross-section of the beam at the sensor surface than in the case of beam walking, this ATR-microscope has another shortcoming.
Thus, in WO 98/34098, as in conventional ATR-spectroscopy, the center angle of the collimated beam is orthogonally incident onto the entrance surface of the ATR-prism. During the angle scan of this collimated beam, however, the angle of incidence at the entrance surface of the prism will be oblique, a typical angle range being, say, 15xc2x0. Therefore, even for an illuminated ATR-sensor area with eliminated beam walking, i.e. a fixed center during angle scan, there will be a variation in the light intensity, or irradiance, with the angle of incidence since the intersection area of the incident collimated beam (of mainly constant beam cross-section area) with the ATR-surface will vary in accordance to Lambert""s Cosine Law. The length of the intersection area thus has a fixed center but the length may be said to xe2x80x9coscillatexe2x80x9d around its center.
For example, if the length of the intersection area is denoted L, the length L=(beam diameter)/cosine(angle of incidence), or, in case section, L=(beam cross-section length)/cosine(angle of incidence). This causes, even if the beam walking has been eliminated, L (and the illuminated area) to increase, and the irradiance to decrease, the larger the angle of incidence at the sensor area. The irradiance is proportional to cosine(angle of incidence), during the angle scan by the following exemplary factors: for angle of incidence scan 63xc2x0 to 77xc2x0: 2.0xc3x97; for angle of incidence scan 62xc2x0 to 82xc2x0: 3.37xc3x97; and for angle of incidence scan 64xc2x0 to 84xc2x0: 4.19xc3x97.
This monotone continuously decreasing light intensity with cosine(angle of incidence) creates a correspondingly sloping TIR-data vs. angle curve. As is readily seen, the reflectance at 77xc2x0 will be about 50% of that at 63xc2x0, and at 84xc2x0 only about 25% of that at 63xc2x0.
Such a substantial decrease in light intensity with increased angle of incidence can not readily be compensated for by computerized data processing like the above-mentioned normalization of the TIR-curve, or at least not without a complex normalization method and software. There is therefore a need for means that overcome the problem of varying light intensity during angular scan in ATR-based microscopy.
Related to ATR-microscopy are other microscopic techniques also based on the evanescent wave phenomenon at total internal reflection without or with evanescent field enhancing metal films or textures, such as, for example, total internal reflection fluorescence (TIRF), total internal reflection phosphorescence, and scattered total internal reflectance (STIR). To overcome the above-mentioned problem of varying light intensity during angular scan would therefore be valuable also in those as well as in other related TIR-techniques.
The present invention overcomes the disadvantages of the prior art TIR-based microscopes, such as the ATR-microscope, and offers additional advantages. In brief, the present invention is based on the concept of varying the intensity of the collimated beam incident on a TIR-sensor surface of fixed center position (i.e. eliminated beam walking) with the angle of incidence, such that lengthwise extension of the illuminated sensor surface area as the angle of incidence is increased, or the effect of such length extension, is counteracted. In this way, the variation of the radiation intensity at the TIR-sensor surface of fixed center position during angular scan may thus be eliminated or at least considerably reduced.
In one aspect, the present invention therefore provides an optical apparatus for total internal reflection (TIR) spectroscopy comprising:
a transparent body having a first entrance surface for incident electromagnetic radiation, a second plane internally reflective surface for reflecting radiation transmitted from the first surface through the transparent body, and a third exit surface through which electromagnetic radiation reflected at the second surface exits the transparent body,
at least one source of electromagnetic radiation,
collimating means for collimating radiation emitted from the at least one source of electromagnetic radiation to at least one beam of collimated electromagnetic radiation,
optical scanning means, arranged between the collimating means and the transparent body, for directing at least part of the collimated electromagnetic radiation to the transparent body so that the radiation is internally reflected at the second surface, and sequentially or continuously scanning the incident angle of the radiation at the second surface over an angular range, the illuminated area or areas having an at least substantially fixed center position, and
at least one detector for detecting electromagnetic radiation exiting from the transparent body.
The apparatus is characterized in that it comprises means for varying the cross-sectional intensity of at least one of (i) the at least one beam of collimated electromagnetic radiation incident on the second plane surface of the transparent body and (ii) the at least one beam of collimated electromagnetic radiation reflected from the second plane surface of the transparent body, in dependence of the incident angle at the second surface during the angular scan, so that variation of the irradiance at the illuminated area or areas of fixed center position of the second plane surface due to varying length extension in the plane of incidence of the illuminated area or areas during the angular scan, or the effect of such variation on the reflected beam or beams, is counteracted.
In one embodiment of the above-mentioned aspect of the present invention, the apparatus is to be used for attenuated total reflection (ATR) spectroscopy and the detector or detectors are arranged to detect electromagnetic radiation totally internally reflected at the second surface and exiting from the third surface of the transparent body.
In another embodiment, the detector or detectors are arranged to detect electromagnetic radiation originating from evanescent wave stimulated fluorescence or phosphorescence of a substance(s) in contact with the second reflective surface of the transparent body.
In still another embodiment, the detector or detectors are arranged to detect electromagnetic radiation originating from scattering of a substance(s) at the second surface of the transparent body.
In yet another embodiment, the detector or detectors are arranged to detect at least two of electromagnetic radiation totally internally reflected at the second surface, electromagnetic radiation originating from evanescent wave stimulated fluorescence or phosphorescence, and electromagnetic radiation originating from scattering at the second surface of the transparent body.
The above mentioned entrance and exit surfaces of the transparent body may optionally be one and the same surface.
In another aspect, the present invention provides an optical apparatus for examining thin layer structures on a surface for differences in respect of optical thickness (and/or refractive index), comprising:
a sensor unit having at least one sensing surface with a number of zones capable of exhibiting thin layer structures of varying optical thickness, particularly as the result of contact with a sample,
at least one source of electromagnetic radiation,
collimating means for collimating radiation emitted from the at least one source of electromagnetic radiation to at least one beam of collimated electromagnetic radiation,
optical means for coupling at least part of the collimated electromagnetic radiation to the sensor unit to illuminate a sensing surface area or areas thereof,
detector means,
means for imaging onto the detector means at least one of (i) radiation internally reflected from the illuminated sensing surface area or areas, (ii) radiation originating from sample on the sensing surface through evanescent wave stimulated fluorescence or phosphorescence, and (iii) radiation originating from scattering from sample at the sensing surface, for detecting the intensities of the radiation reflected or originating, respectively, from the different parts of the illuminated area or areas,
means for sequentially or continuously scanning the radiation incident at the optical coupling means and at the illuminated area or areas of the at least one sensing surface over a range of incident angles, the illuminated area or areas having an at least substantially fixed center position,
means for determining each angle of incidence of electromagnetic radiation impinging on the at least one sensing surface, and
evaluation means for determining from the relationship between detected intensity of radiation imaged on the detector means and incident angle of the radiation reflected at the sensing surface or surfaces, the optical thickness of each sensing surface zone to thereby produce a (morphometric) image of the optical thickness of the at least one sensing surface.
The apparatus is characterized in that it comprises means for varying the cross-sectional intensity of at least one of (i) the at least one beam of collimated electromagnetic radiation incident on the at least one sensing surface area of the sensor unit, and (ii) the at least one beam of collimated electromagnetic radiation reflected from the at least one sensing surface area, in dependence of the incident angle at the sensing surface or surfaces during the angular scan, so that the variation of the irradiance in the illuminated area or areas of fixed center position of the sensing surface or surfaces due to varying length extension in the plane of incidence of the illuminated area or areas during the angular scan, or the effect of such variation on the reflected beam or beams, is counteracted.
In still another aspect, the present invention provides a method of performing total internal reflection based spectroscopy, which method comprises:
irradiating at least one area of a plane surface of a transparent body with at least one collimated beam of electromagnetic radiation so that the radiation is totally internally reflected at the surface,
imaging onto a respective two-dimensional detector array at least one of (i) radiation internally reflected from the illuminated area or areas, (ii) fluorescent or phosphorescent radiation from the illuminated area or areas caused by evanescent wave stimulation, and (iii) radiation originating from evanescent wave stimulated scattering at the illuminated area or areas,
sequentially or continuously scanning the incident angle over an angular range, the illuminated area or areas having an at least substantially fixed center position during angular scan,
measuring at at least a number of incident angles of the radiation reflected at the plane surface of the transparent body, the intensities of the radiation imaged on different parts of the detector array (D), and
determining from the detected radiation intensities at the different incident angles at the transparent body surface, at least one of an optical thickness image, a refractive index image, a surface concentration image of the surface, and the variation of such images with time.
The method is characterized in that it comprises continuously varying the cross-sectional intensity of at least one of (i) the at least one beam of collimated electromagnetic radiation incident on the plane surface of the transparent body, and (ii) the at least one beam of collimated electromagnetic radiation reflected from the plane surface of the transparent body, in dependence of the incident angle at the surface during the angular scan to reduce variations of the intensity of radiation in the illuminated area or areas of fixed center position of the surface due to varying length extension in the plane of incidence of the illuminated surface area or areas of fixed center position during the angular scan, or the effect of such variation with time on the reflected beam or beams.
In one embodiment, the method is used for kinetic studies of binding events at a sensor surface.
In a particular embodiment of the above method aspect of the invention, ATR-spectroscopy is performed, i.e. the radiation internally reflected from the illuminated area or areas is imaged onto the detector array.
In preferred embodiments of the above apparatus and method aspects of the invention, variations of the intensity of radiation in the illuminated area(s) of the surface of the transparent body caused by varying length extension of the illuminated area(s) during the angular scan are counteracted by varying the length extension in the plane of incidence of the incident beam (or beams) of collimated electromagnetic radiation in dependence of the incident angle during the angular scan, and more particularly such that the beam cross sectional area is decreased as the angle of incidence at the surface is increased.
Optionally, the apparatuses and method, respectively, of the present invention are arranged to permit use of at least two different wavelengths.
The above and other aspects of the invention will be evident upon reference to the attached drawings and the following detailed description.